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Many things might happen in the body when a titanium implant is inserted into bone. Examples are activation of the immune system and imbalance between bone formation and bone resorption, which might lead to damaged bone around the implant and at worse, loosening of the implant. Bisphosphonates, BP’s, is a class of drugs that is able to decrease the osteoclast (bone resorption cell) activity and thereby strengthen the bone. FibMat2.0 is a fibrinogen matrix and consists of a thin protein layer which can be applied on an implant and act as a local drug delivery system. The work in this thesis was divided into two parts where aim of the first part was to study FibMat2.0 with integrated BP’s, and their effect in the presence of blood. The aim for the second part was to determine whether it was possible to incorporate antithrombotic drugs into the fibrinogen matrix. No detection method for the amount of drugs incorporated into the fibrinogen matrix was used but the fact that the drugs gave effect was verifying that it is possible to integrate other drugs than BP’s into FibMat2.0. Methods that have been used in the experiments in presence of blood are imaging of coagulation, fluorescence microscopy and cone-and-plate. For the first part, the results showed that surfaces incubated with fibrinogen and fibrinogen with integrated BP’s act alike in regard to coagulation and platelet adhesion. Compared to titanium, which is known to be a biocompatible material, the surfaces with fibrinogen and fibrinogen with BP’s behave similar in regard to platelet adhesion. When it comes to coagulation, the surfaces coated with fibrinogen with or without an addition of BP’s have shown a longer coagulation time compared to the clean titanium surface. For the second part, some conclusions have been drawn according to the results. Heparin and hirudin have shown anticoagulant effects when integrated in the matrix. The platelet inhibitor cangrelor seemed to have better effect when added in blood and incubated compared to incubation with the platelet inhibitor on the surface before incubation in blood. Finally, when combining heparin and cangrelor, very clear differences in regard to formation of fibrin network could be seen. It seems promising to be able to load different kind of drugs in FibMat2.0.

Multimerin 1 (human: MMRN1, mouse: Mmrn1) is a large homopolymeric glycoprotein that is synthesized and stored by platelets and endothelial cells until activation-induced release. MMRN1 is able to support platelet adhesion through mechanisms involving von Willebrand factor (VWF) and glycoprotein (GP)Ibα, and β3 integrins on activated platelets, and it enhances platelet adhesion to fibrillar collagen, potentially by binding to putative MMRN1-specific GPAGPOGPX (where O is hydroxyproline and X is valine or glutamine) motifs in fibrillar collagens. Using mice with and without selective Mmrn1 deficiency, the goals of this thesis were: 1) further characterize the ability of Mmrn1 to enhance platelet adhesion to collagen, 2) explore the role of fluid shear stress in the ability of Mmrn1 to enhance platelet adhesion, and 3) test the specificity of the GPAGPOGPX motif for Mmrn1 and the ability of GPAGPOGPX to support or enhance platelet adhesion. Mmrn1-deficient (Mmrn1-/-) mouse platelets showed impaired aggregate formation on fibrillar collagen surfaces under high (1500 s-1) and low (300 s-1) shear flow compared to wild-type (Mmrn1+/+) mouse platelets, which was due to reduced initial adhesion and a slower rate of platelet accumulation onto collagen surfaces. Similarly, Mmrn1-/- platelets formed smaller aggregates on immobilized recombinant (r)Vwf surfaces compared to wild-type platelets, and Mmrn1-/- platelets had impaired adhesion and aggregate formation on immobilized murine fibrinogen, but not fibrin, when platelets were pre-activated to release Mmrn1. Type I fibrillar collagen was found to contain a variant of the GPAGPOGPX motif (GPAGPOGPI), and GPAGPOGPX motifs supported adhesion of wild-type, but not Mmrn1-/-, platelets. When presented with the VWF-binding GPRGQOGVMGFO motif and the integrin α2β1-binding GFOGER motif present in fibrillar collagens, the GPAGPOGPX motifs synergistically enhanced platelet adhesion. These findings expand upon the known adhesive functions of platelet multimerin 1 and update knowledge of the motifs that support platelet adhesion to fibrillar collagens. / Dissertation / Doctor of Philosophy (Medical Science)

Stent is one of the coronary angioplasty techniques that expands the narrowed coronary arteries due to the accumulation of fat, cholesterol and other substances in the lumen of the arteries. The major complication of stent is restenosis. Current development of drug-eluting stents shows successfully reduce the occurrence of restenosis. Other than using drugs, electrostatic self assembled (ESAd) thin films may be the potential candidates to prevent restenosis. ESA is a process to fabricate thin films bases on the electrostatic attraction between two oppositely charges. We used this technique to fabricate four PVP films and four PEI films. All films were examined by XPS and AFM. XPS data showed our coatings were successfully fabricated on substrates. AFM images revealed PVP coating was uniform, but PEI coatings had different morphologies due to diffusion and pH during the process. Three preliminary hemocompatibility testes were performed to evaluate the hemocompatibility of the coatings. Platelet adhesion study showed the thin films inhibited platelet adhesion. All thin films were able to inhibit coagulation and were less cytotoxic. The studies suggested the ESA films were potentially hemocompatible. / Master of Science

According to the World Health Organization (WHO), Cardiovascular Disease (CVD) is the leading cause of death in the world. Biomechanics and fluid dynamics of blood flow play an important role in CVD mediation. Shear stress plays a major role in platelet-substrate interactions and thrombus formation and growth in blood flow, where under both pathological and physiological conditions platelet adhesion and accumulation occur. In this study, a three-dimensional dynamic model of platelet-rich thrombus growth in stenosed vessels using computational fluid dynamics (CFD) methods is introduced. Platelet adhesion, aggregation and activation kinetics are modeled by solving mass transport equations for blood components involved in thrombosis. The model was first verified under three different shear conditions and at two heparin levels. Three-dimensional simulations were then carried out to evaluate the performance of the model for severely damaged (stripped) aortas with mild and severe stenosis degrees. For these cases, linear shear-dependent functions were developed for platelet-surface and platelet-platelet adhesion rates. It was confirmed that the platelet adhesion rate is not only a function of Reynolds number (or wall shear rate) but also the stenosis severity of the vessel. General correlations for adhesion rates of platelets as functions of stenosis and Reynolds number were obtained based on these cases. The model was applied to different experimental systems and shown to agree well with measured platelet deposition. Then, the Arbitrary Lagrangian Eulerian (ALE) formulation was used to model dynamic growth by including geometry change in the simulation procedure. The wall boundaries were discretely moved based on the amount of platelet deposition that occurs on the vessel wall. To emulate the dynamic behavior of platelet adhesion kinetics during thrombus growth, the validated model for platelet adhesion, which calculates platelet-surface adhesion rates as a function of stenosis severity and Reynolds number, was applied to the model. The model successfully predicts the nonlinear growth of thrombi in the stenosed area. These simulations provide a useful guide to understand the effect of growing thrombus on platelet deposition rate, platelet activation kinetics and occurrence of thromboembolism (TE) in highly stenosed arteries. / Ph. D.

Streptococcus sanguinis is a leading cause of infective endocarditis. Bacterial adhesion to platelets is likely important in the pathogenesis of infective endocarditis. Bacterial cell wall-anchored (Cwa) proteins may mediate this adhesion. To begin to test this hypothesis, S. sanguinis adhesion to platelets was examined in vitro. The requirement of 12 Cwa proteins for S. sanguinis-platelet adhesion was individually assessed, measuring adhesion of purified platelets to polystyrene wells coated with S. sanguinis strain SK36 or 12 isogenic Cwa protein mutants. Significantly fewer platelets adhered to wells coated with one mutant strain, VT1614. However, results of a whole-cell enzyme-linked immunosorbent assay (ELISA) showed that 8 mutants, including VT1614, adhered in significantly lower numbers to wells than did SK36. After accounting for unequal bacterial numbers, we determined there was no significant difference in platelet adhesion among the strains. This suggests that none of the Cwa proteins examined were required for S. sanguinis-platelet adhesion.

Platelets are crucial for prevention of blood loss after vessel injury. Platelet adhesion to disrupted vessel walls is mediated by receptors such as the GPIb-IX-V complex that binds von Willebrand factor and the collagen-binding integrin α2β1. Also cross-linking of platelets, mediated by αIIbβ3 that binds to fibrinogen, results in platelet aggregation that further contributes to hemostasis. Platelets are also important pathophysiologically because of their role in thrombus formation following atherosclerotic plaque rupture. Pharmacological treatments aimed to prevent such events include use of platelet inhibitors such as acetylsalicylic acid (ASA) and clopidogrel. Despite the presence of several different platelet function assays, no one has so far been considered useful for clinical evaluation of the effect of anti-platelet treatment. The aim of this thesis was to evaluate possible applications in experimental as well as in clinical research for a platelet adhesion assay performed during static conditions. In principle, platelets in plasma are allowed to attach to protein coated microplates. Adhered platelets are then detected by induction of an enzymatic reaction followed by spectrophotometric measurements of the developed product. Our results show that the platelet adhesion assay is able to detect experimentally induced activation as well as inhibition of platelets. The assay also seems useful for investigation of synergistically induced platelet activation, especially when the coated surface consists of albumin. This is exemplified by the combination of lysophosphatidic acid and adrenaline, which induced a synergistically increased platelet adhesion to albumin that was dependent on αIIbβ3-receptors and on the secretion of ADP. Furthermore, secretion of ADP as well as TXA2 seems to contribute to several adhesive reactions investigated with this assay. The dependence on secretion, together with results showing that adhesion to collagen and fibrinogen is dependent on α2β1- and αIIbβ3-receptors respectively, indicate that the adhesive interactions occurring in the assay is in accordance with the general knowledge about platelet function. Regarding clinical applications, we found that platelet adhesion was increased for patients with essential thrombocythemia (ET) compared to controls. This is in line with the in vivo function of ET-platelets since a common complication for ET-patients is thrombosis. Furthermore, the assay was able to detect effects of treatment with clopidogrel in patients with unstable angina. To some extent it also measured the effects of ASA-treatment. In conclusion, our results suggest that the assay is suitable for experimental research and that further studies should be performed aimed at developing the assay into a clinically useful device.

Arterial thrombus forms from the capture and accumulation of circulating platelets on a stenosis. As the thrombus grows, the lumen becomes further stenotic producing very high shear rates as the blood velocities increase through the narrowed cross-section. This study explores the molecular binding conditions that may occur under these pathologic shear conditions where circulating platelets must adhere quickly and with strong bonds. Platelets binding in an arterial stenosis of >75% are subject to drag forces exceeding 10,000 pN. This force can be balanced by 100 simultaneous GPIb-vWFA1 bonds of 100 pN each. The number and density of GPIb on platelets is sufficiently high; however, platelet capture under high shear would require the density of A1 receptors to be increased to over 416 per square micron. A computational model is used to determine platelet capture as a function of shear rate, surface receptor density, surface contact and kinetic binding rate. A1 density could be increased by a combination of vWF events of: i) plasma vWF attach to the thrombus surface and elongate under shear; ii) the elongated vWF strands create a net with 3-D pockets; and iii) additional vWF is released from mural platelets by activation under shear. With all three events, A1 density matches the existing high GPIbα densities to provide sufficient multivalency for capture at 100,000 s-1 with greater than 170 bonds per platelet. If the on-rate is greater than 108 M-1s-1, then a platelet could be captured within 15 microseconds, the amount of time available to form bonds before the platelet is swept away. This mechanism of platelet capture allows for the rapid platelet accumulation in atherothombosis seen clinically and in high shear experiments.

The hemocompatibility of crystalline Silicon Carbide (SiC), in its cubic form (i.e., 3C-SiC), has been evaluated and compared to Silicon (Si), the leading material in biosensing applications. Silicon carbide (SiC) is a hard, chemically robust material, very well suited for harsh environment applications, and has been suggested to have very good biocompatibility. Additionally, SiC in its amorphous form, has been used as a coating for medical implantable devices such as bone prosthetics and cardiovascular stents. However, assessment of single crystal 3C-SiC for cardiovascular applications has not been reported. In this research we have studied the interactions of single crystal 3C-SiC with platelets and human microvascular endothelial cell (HMVEC) to assess the degree of hemocompatibility of 3C-SiC. The more hemocompatible a material is, the less platelet adhesion would be expected. Using fluorescence microscopy higher platelet adhesion was statistically observed on Si than on SiC. In addition 3C-SiC surfaces showed less platelet reactivity, measured by the degree of platelet adhesion, aggregation and activation, with mostly circular morphology of adhered platelets while Si showed an elevated presence of non-activated (Circular) platelet clumps. Additionally, HMVEC proliferation assessment suggest that 3C-SiC performs comparably to high attachment culture wells with enhanced proliferation, without affecting cell morphology. These results suggest that 3C-SiC is a promising candidate for applications in the blood stream due to its low thrombogenic characteristics and good hemocompatibility.

Understanding the differential bonding mechanics underlying bleeding disorders is of crucial importance to human health. In this research insight is provided into how four of these bleeding disorders (each with somewhat similar clinical characteristics), work at the molecular bond level. The bleeding diseases studied here can result from defects in the platelet glycoprotein (GP) Ibα the von Willebrand factor (vWF) molecule, or the ADAMTS-13 enzyme. Types 2B and 2M von Willebrand Disease (VWD) result in excess bleeding, yet type 2B has increased binding affinity between platelet GPIbα and vWF, while type 2M has decreased binding affinity between these two molecules. Platelet type VWD (pt-VWD) causes mutations in the GPIbα molecule and has similar characteristics to type 2B VWD. Further, in thrombotic thrombocytopenic purpura, bleeding results when there is a lack of active ADAMTS-13 enzyme. Each disease results in patient bleeding, but due to different mechanisms. This dissertation will explore the bonding mechanics between GPIbα and vWF and how they are altered in each disease state. To observe the GPIbα-vWF bonding mechanics, rolling velocities, transient tethering lifetimes, and tether frequency were determined using a parallel plate flow chamber. Data from these experiments suggest that wt-wt interactions are force dependent and have biphasic catch-slip bonding behavior. The data show that the shear stress at which the maximum mean stop time occurs differs between gain-of-function and loss-of-function mutations. Using similar methods, we study the changes resulting from pt-VWD mutations in GPIbα, and find that the catch bond seen for wt-wt interactions is lost for these mutations. Further, the data suggest that interactions with gain-of-function GPIbα mutations may be transport rather than force dependent. Finally, how the GPIbα-vWF tether bond changes for thrombotic thrombocytopenic purpura was also investigated to show that the bond lifetime in the absence of the enzyme is increased presenting a possible rationale for why bleeding occurs in this disease. Overall, the data show how the bonding mechanics of the GPIbα-vWF tether bond differ in four bleeding diseases. Further, these observations offer potential explanations for how these changes in the bonding mechanism may play a role in the observed patient bleeding.